The principles underlying the coordinated macromolecular interactions which culminate in the assembly of infectious viruses are not well understood. A generally accepted model postulates that small macromolecular oligomers form a nucleating template that is required for initiation of particle formation. Moreover, it is evident in many cases that viral morphogenesis continues after completion of a spherically closed shell. These two concepts are the basis for the specific aims of this proposal. Our experimental strategy integrates electron cryomicroscopy, image processing, X-ray crystallography, and molecular modeling to explore the specific assembly mechanisms of 3 families of icosahedral viruses, nodaviruses, tetraviruses and sobemoviruses. Cryo-EM is a particularly powerful technique in this regard since dynamic states and short-lived intermediates can be trapped and imaged in the frozen-hydrated state. Three-dimensional density maps can be combined with subunit atomic structures provided by X-ray crystallography in order to build pseudo-atomic models. Our proposal is supported by 7 publications over the last 5 years. We plan to use in vitro assembly of a number of mutants of the nodavirus flockhouse virus to define how RNA:protein interactions control assembly. Time-resolved cryo- EM will be used to explore the dramatic conformational changes that occur when the tetravirus NoV procapsid is converted to the mature capsid. To examine a pseudo-atomic model that we have proposed for the procapsid, we plan to determine a 3D reconstruction at better than 10Angstroms resolution. Comparable resolution will be necessary to understand the structural rearrangements that occur during the contraction of the sobemovirus RYMV during chelation of divalent metal ions and to gain a better understanding of RNA:protein interactions in the nodavirus PAV. These experiments will reveal details about the ways in which virus assembly is initiated by nucleating templates and the ways in which coordinated transformations between intermediates culminate in mature infectious particles. Our results will contribute to our general understanding of the mechanistic details of virus assembly.